Method of proliferating human hepatocytes and method for obtaining human hepatocytes

ABSTRACT

Transplanting human hepatocytes into a liver of an immunodeficient hepatopathy mouse, and then feeding the mouse transplanted with the human hepatocytes under such a condition as being protected from the attack by human complement produced by the human hepatocytes thereby proliferating the transplanted human hepatocytes in the mouse liver. Further, Obtaining human hepatocytes in large scale by repeating the above steps using the proliferated human hepatocytes.

TECHNICAL FIELD

The present invention relates to a method for proliferating humanhepatocytes in a mouse body, a chimeric mouse carrying human hepatocytesin the liver thereof, a method for obtaining human hepatocytes from saidchimeric mouse, and human hepatocytes obtained thereby. Humanhepatocytes obtained from said chimeric mouse may be used as a materialof a hepatocyte kit or an extracorporeal artificial liver.

BACKGROUND ART

A liver has 500 or more types of various specific functions. Forexample, major functions of a liver are plasma protein synthesis andexcretion, blood sugar control by gluconeogenesis and glycogenmetabolism, lipogenesis, ureogenesis, bile synthesis and excretion,detoxication and so on.

Most substances incorporated into a body are mainly metabolized in aliver. In the field of pharmaceutical development, what type ofmetabolism pharmaceutical candidate substances will be received in aliver and what type of effect is given to a liver or other organs andtissues are essential data. Further, many chemical substances have beensynthesized and discharged into environment up to now. To elucidate whatkind of effect these substances have exerted individually or incombination to a human body is socially very important. Toxicity test onliver functions are essential for evaluation of the effects of suchchemical substances to a human body.

Mice, rats, rabbits, dogs, monkeys, etc are used at present for safetytests and drug metabolism tests of chemical substances includingpharmaceutical candidate substances. Especially in pharmaceuticaldevelopment, toxicological tests and safety tests using animals arecompulsive before entering phase I study for human; therefore, longperiod and efforts as well as huge costs are required in these tests.

However, there is no guarantee that data obtained by these animalexperiments can be applicable to human. In fact, many cases are knownwherein a substance not recognized toxicity in animal experimentsexhibited toxicity in human or vice versa. Consequently, it is expectedup to now that development of many pharmaceutical candidate substancesis aborted after entering in phase I study on human, and besidesexpected that there is many case wherein although a substance hasactually no toxicity in human and the development thereof is abortedbefore entering a clinical trial due to exhibiting strong toxicity inanimal experiments.

This may be caused by difference in metabolic functions in a human liverand metabolic functions in livers of mice and rats. Recently, in vitrometabolic tests and toxicity tests using human hepatocytes have beenperformed. However, amount of livers from brain death patients whichwere not used for transplantation and amount of human hepatocytesobtained from hepatectomy in tumor excision are far fewer than demanded.Consequently, development of technology for human hepatocyteproliferation is essential for pharmaceutical development.

Necessity of high amount of human hepatocytes is very much alike in anextracorporeal artificial liver. The artificial liver is medical deviceacting liver function artificially. It is vigorously in progress todevelop a hybrid artificial liver combining with the artificial actionbased on physicochemical principle such as adsorption, dialysis andfiltration, along with biological actions using perfusion of an excisedliver and liver tissue. In such a development of an artificial liver, itis essential to improve membrane and circuit for enhancingphysicochemical function, along with to supply high amount ofhepatocytes applicable to human use.

However, in a case of human hepatocytes, it has been consideredimpossible to serially subcultivate primary cells, which are separatedfrom matured individual. Namely, the matured hepatocytes with adhesiondependency are largely damaged when cells are detached from culturesubstrate for subcultivation operation and are difficult to re-adhere toculture substrate. Contrary to that, the present inventors of thisapplication have invented the methods for proliferating hepatocytes,wherein small hepatocytes having clonal proliferative ability wereisolated from normal hepatocytes separated from human liver, andfurther, it was carried out to primarily culture the small hepatocytesand then to subculture the cultured hepatocytes; and which were grantedpatents (JP-A-08-112092, JP No.3266766. U.S. Pat. No. 6,004,810;JP-A-10-179148, JP No.3211941; JP-A-07-274951, JP No. 3157984; andJP-A-09-313172, JP No.3014322).

Although the methods of those patented inventions provide new techniqueto obtain human hepatocytes in large scale by in vitro proliferation ofhepatocytes, there remains a problem of diminishing some liver functionsduring long period subculture. Therefore, these cells obtained by theabove-described methods are useful, for example, for a screening systemof medical drug for maintaining liver function, or for a testing systemof toxicity and efficacy of medical drugs in terms of certain functionsremained after long period subculture, however, these cells areinsufficient to use as substitution of human liver function, or for amaterial of a hybrid-type artificial liver.

As a measure to solve the above problems in proliferation of hepatocytesin vitro, a method for proliferating human hepatocytes in animal body(in vivo) has been proposed.

For example, Heckel, et al. prepared the albumin-urokinase-typeplasminogen activator transgenic mouse (uPA-Tg mouse). In this mouse, asthe urokinase plasminogen activator (uPA) gene is attached to anenhancer and a promoter of albumin, uPA protein is specificallyexpressed in a liver (Heckel J L, Sandgren E P, Degen J L, Palmiter R D,and Brinser R L; Neonatal bleeding in transgenic mice expressingurokinase-type plasminogen activator. Cell 62:447-456, 1990). The colorof hepatocytes of said mouse looks white by naked eye due to injury byuPA protein, and some mice die by bleeding or liver failure. It has beenknown that when normal hepatocytes of lacZ transgenic mouse weretransplanted into a spleen of the above-mentioned mouse, thetransplanted hepatocytes are attached to the liver and proliferated, andfinally the recipient hepatocytes are replaced by the donor hepatocytes(Rhim J A, Sandgren E P, Degen J L, Palmiter R D, and Brinster R L;Replacement of diseased mouse liver by hepatic cell transplantation.Science 263:1149-1152, 1994). Further, Rhim, et al. prepared theuPA-Tg/NUDE mouse by mating between the uPA-Tg mouse and a NUDE mousewhich has no T-cell function due to hereditary deletion of thymus. Amouse having rat hepatocytes was prepared by transplantation of rathepatocytes into the uPA-Tg/NUDE mouse (Rhim J A, Sandgren E P, PalmiterR D and Brinster R L; Complete reconstitution of mouse liver withxenogeneic hepatocytes. Proc. Natl. Acad. Sci. USA 92:4942-4946, 1995).However, there is no report on a chimeric mouse carrying humanhepatocytes using said mouse.

Dandri, et al. prepared the uPA-Tg/Rag2 mouse by mating the uPA-Tg mouseand Rag2 mouse which is a knockout mouse having an immunodeficientcharacteristics. It has been reported that 15% of the mouse liver wasreplaced by human hepatocytes when human hepatocytes obtained from ahuman liver were transplanted into a liver of the uPA-Tg(±)/Rag2 mouse.Further they were succeeded in in vivo infection with hepatitis B virusto said mouse (Dandri M, Burda M R, Torok B, Pollok J M, Iwanska A,Sommer G, Rogiers X, Rogler C E, Gupta S, Will H, Greten H, and PetersenJ; Repopulation of mouse liver with human hepatocytes and in vivoinfection with hepatitis B virus. Hepatology 33:981-988, 2001).

Also, the inventors of this present invention have disclosed in a priorpatent application (JP-A-2002-45087) that in a chimeric mouse producedby transplantation of human hepatocytes into an uPA-Tg/SCID mouseobtained by mating the uPA-Tg mouse and SCID mouse which is animmunodeficient mouse, the transplanted human hepatocytes havesubstantially taken place functions of a mouse liver. In this chimericmouse of the prior invention, mouse hepatocytes have been dysfunctioneddue to expression of uPA gene, and thus the liver function is maintainedby the transplanted human hepatocytes. Therefore, said chimeric mouse isquite useful as an experimental animal for evaluating toxicity andefficacy of test substances because the in vivo function of transplantedhuman hepatocytes may be evaluated precisely. However, the chimericmouse of the prior invention cannot live 50 days or longer aftertransplantation of human hepatocytes. Also, due to proliferationnormalized mouse hepatocytes in the course of growing, proliferationefficiency of transplanted human hepatocytes was low and replacementratio of human hepatocytes remained about 50%.

Above finding has also been supported by a recent report published byMercer, et al. (Mercer D F, Schiller D E, Eliiott J F, Douglus D F, HaoC, Ricnfret A, Addison W R, Fischer K P, Churchill T A, Lakey J R T,Tyrrell D L J and Keteman N M; Hepatitis C virus replication in micewith chimeric human livers. Repopulation of mouse liver with humanhepatocytes and in vivo infection with hepatitis B virus. NatureMedicine 7:927-933, 2001). Mercer, et al. have reported that theuPA-Tg/SCID mouse, prepared by similar procedures as described by theinventors of this present application, was transplanted with thawedhuman hepatocytes which had been stored in a frozen state; as theresults, less than 2 mg/ml of human albumin was detected in mouse serum(equivalent to approximately less than about 1 mg/ml in blood) implyingthat about 50% of liver have been replaced by human hepatocytes.

As described above, a chimeric mouse which is prepared by atransplantation of human hepatocytes into an immunodeficient hepatopathymouse (uPA-Tg/ SCID mouse) was insufficient as means to proliferatetransplanted human hepatocytes in large scale in a mouse, even though achimeric mouse itself had usefulness (for example, for in vivo testingof toxicity or efficacy to human hepatocytes).

Moreover, the chimeric mouse transplanted with human hepatocytes can notlive for long period of time; and since mouse hepatocytes proliferate ina course of its growing, availability of such a chimeric mouse as an invivo evaluation system of toxicity or efficacy against human hepatocyteshas been limited.

Scope of the present invention is to provide, as a method forproliferating human hepatocytes, an improved method for proliferatinghuman hepatocytes sufficiently in a mouse body.

Also, to provide a method for separation and recovery of humanhepatocytes propagated in a mouse body by extension of life time is alsoincluded in the scope of the present invention.

Further, the scope includes to provide a method for obtaining a largenumber of chimeric mice carrying human hepatocytes with certainspecifications by means of transplantation of separated humanhepatocytes into plural mice and proliferation in mice bodies, and amethod for obtaining human hepatocytes with certain specificationsseparated from said mouse in large scale.

Furthermore, to provide a method for application of separated humanhepatocytes is also included in the scope.

In addition, the scope of the present application is to provide a usefulmonoclonal antibody useful to operate the above-described eachinvention, and a new hybridoma cell line producing said monoclonalantibody.

DISCLOSURE OF INVENTION

The present application provides, as the 1st invention to solve theabove-described problems, method for proliferating human hepatocytes,which comprises transplanting human hepatocytes into a liver of animmunodeficient hepatopathy mouse, and then feeding the mousetransplanted with the human hepatocytes under such a condition as beingprotected from the attack by human complement produced by the humanhepatocytes thereby proliferating the transplanted human hepatocytes inthe mouse liver.

In a preferred embodiment of the method of the 1st invention, thecondition of being protected from the attack by human complement is atleast one of the following (a) and (b):

-   -   (a) the mouse transplanted with the human hepatocytes is        administered at least once with a complement inhibitor;    -   (b) a progeniture mouse obtained by mating between an        immunodeficient hepatopathy mouse and a decay-accelerating        factor (DAF/CD55) transgenic mouse is utilized as the        immunodeficient hepatopathy mouse.

Also, in the method of the 1st invention, a preferred embodiment is thatthe immunodeficient hepatopathy mouse is the progeniture mouse obtainedby mating between a genetically immunodeficient mouse and a geneticallyhepatopathy mouse. Additionally, a preferred embodiment of the method isthat the progeniture mouse is a hemizygous immunodeficient hepatopathymouse, and this hemizygous immunodeficient hepatopathy mouse isadministered with a hepatocyte growth inhibitor and then the humanhepatocytes are transplanted therein.

Further, in the method of the 1st invention and preferred embodimentsthereof described above, it is another preferred embodiment that theimmunodeficient hepatopathy mouse transplanted with the humanhepatocytes is administered with an anti-mouse Fas antibody.

Also, in the 1st invention the following each method is also a preferredembodiment, wherein the human hepatocytes to be transplanted into theimmunodeficient hepatopathy mouse are proliferative human hepatocytes,and the proliferative human hepatocytes are human hepatocytes recognizedby a monoclonal antibody which specifically recognizes human hepatocyteswhich proliferate, and the monoclonal antibody is one produced fromMouse-Mouse hybridoma K8223 (FERM BP-8334).

The present application provides, as the 2nd invention, a method forproliferating human hepatocytes in large scale, which comprises thefollowing steps (1) to (3), and the steps (2) and (3) are repeated atleast once;

-   -   (1) a step comprising transplanting human hepatocytes into a        liver of an immunodeficient hepatopathy mouse, and then feeding        the mouse transplanted with human hepatocytes under such a        condition as being protected from the attack by human complement        produced by the human hepatocytes thereby proliferating the        transplanted human hepatocytes in the mouse liver;    -   (2) a step isolating the proliferated human hepatocytes from the        mouse liver; and    -   (3) a step comprising transplanting the human hepatocytes        isolated from the mouse liver into the liver of an        immunodeficient hepatopathy mouse, and then feeding the mouse        transplanted with the human hepatocytes for not shorter than 50        days under such a condition as being protected from the attack        by human complement produced by the human hepatocytes.

In preferred embodiments of the method of the 2nd invention, thecondition of being protected from the attack by human complement in thestep (1) and/or the step (3) is at least one of the following (a) and(b):

-   -   (a) the mouse transplanted with human hepatocytes is        administered at least once with a complement inhibitor;    -   (b) a progeniture mouse obtained by mating between an        immunodeficient hepatopathy mouse and a decay-accelerating        factor (DAF/CD55) transgenic mouse is utilized as the        immunodeficient hepatopathy mouse.

Also, in the 2nd invention, each of the following method is preferable,wherein the immunodeficient hepatopathy mouse in said steps (1) and/or(3) is the progeniture mouse obtained by mating between a geneticallyimmunodeficient mouse and a genetically hepatopathy mouse; theprogeniture mouse is a hemizygous immunodeficient hepatopathy mouse; andsaid hemizygous immunodeficient hepatopathy mouse is administered with ahepatocyte growth inhibitor and then human hepatocytes are transplantedtherein.

Further, in the method of the 2nd invention and its preferredembodiments, another preferred embodiment is the immunodeficienthepatopathy mouse transplanted with human hepatocytes in said steps (1)and/or (3) is administered with an anti-mouse Fas antibody.

Also, in the 2nd invention, there are also preferred embodimentsrespectively, wherein the human hepatocytes to be transplanted into theimmunodeficient hepatopathy mouse in the step (1) and/or the step (3)are proliferative human hepatocytes; the proliferative human hepatocytesare human hepatocytes recognized by a monoclonal antibody whichspecifically recognizes human hepatocytes which proliferate with formingcolony; and the monoclonal antibody is one produced from Mouse-Mousehybridoma K8223 (FERM BP-8334).

Furthermore, in the method of the 2nd invention, it is a preferredembodiment that only human hepatocytes are substantially isolated instep (2) by at least one of the following procedures (a) and (b);

-   -   (a) to treat a liver tissue separated from the mouse liver with        collagenase; and    -   (b) to isolate cells being recognized by a monoclonal antibody        which specifically recognizes human hepatocytes but not        recognizes non-human hepatocytes. In this embodiment, a more        preferable embodiment is that the monoclonal antibody is one        produced by Mouse-Mouse hybridoma K8216 (FERM BP-8333).

The present application provides, as the 3rd invention, a chimeric mousecarrying in the liver human hepatocytes proliferated by theabove-described methods of the 1st invention or the 2nd invention.

Another preferred embodiment is that in the chimeric mouse of the 3rdinvention, the proliferated human hepatocytes make up not less than 70%of the cells in the liver, and/or said mouse has human-type P450activity.

The present application provides, as the 4th invention, a method forobtaining human hepatocytes comprises isolating the human hepatocytesfrom the liver of the chimeric mouse of the above-described 3rdinvention.

Furthermore, in the method of the 4th invention, a preferred embodimentis that only human hepatocytes are substantially isolated by at leastone of the following procedures (a) and (b);

-   -   (a) to treat a liver tissue separated from the mouse liver with        collagenase; and    -   (b) to isolate cells being recognized by a monoclonal antibody        which specifically recognizes human hepatocytes but not        recognizes non-human hepatocytes. And in this embodiment, a more        preferable embodiment is that the monoclonal antibody is one        produced from Mouse-Mouse hybridoma K8216 (FERM BP-8333).

The present application provides, as the 5th invention, the humanhepatocytes obtained by the above-described method of theabove-described 4th invention.

Further, the present application provides, as the 6th invention, acellular kit containing the human hepatocytes of the above-described 5thinvention.

Furthermore, the present application provides, as the 7th invention, ahybrid-type artificial liver filled with the human hepatocytes of theabove-described 5th invention.

Furthermore, the present application provides, as the 8th invention,monoclonal antibody which specifically recognizes human hepatocytes butnot recognize non-human hepatocytes. An example of the 8th invention isthe monoclonal antibody produced from Mouse-Mouse hybridoma K8216 (FERMBP-8333).

Furthermore, the present application provides, as the 9th invention, amethod for testing pharmaceutical kinetics or toxicity of a candidatesubstance, which comprises systemically administering the substance intothe chimeric mouse of the above-described 3rd invention.

Embodiments, terms and concept described in the above each inventionwill be defined more in detail in the sections of Best Mode for CarryingOut the Present Invention or Examples. Various techniques required foroperating the present invention may be available easily and preciselyaccording to the known literature except for the techniques expresslyprovided with references, by those skilled in the art. For example,techniques of genetic engineering and molecular biology used in thepresent invention are described in the following literature; Sambrookand Maniatis, in Molecular Cloning-A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York, 1989; Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1995et al.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is test result on the effect of a complement inhibitor on humanalbumin concentration.

FIG. 2 is measuring result of blood albumin concentration in micetransplanted with human liver parenchymal cells or small hepatocytes.

FIG. 3 is test result on the effect of a complement inhibitor on micebody weight.

FIG. 4 is comparison on human complement (hC3a) concentrations between acomplement inhibitor is administered and not administered in chimericmouse.

FIG. 5 is a graph showing correlations between human albuminconcentration in mice blood and replacement rate by human hepatocytes,along with liver function.

FIG. 6 shows images of immunostaining by a human specific cytokeratin8/18 antibody.

FIG. 7 shows magnified images of immunostaining by a human specificcytokeratin 8/18 antibody.

FIG. 8 is Western blotting analysis result of human specific CYP2C9 inmicrosomes from mouse liver transplanted with human hepatocytes.

FIG. 9 is a graph showing metabolic activity of diclofenac in mouse ordonor liver microsomes.

FIG. 10 is a graph showing expression amount of six types of human P450isozymes in a chimeric mouse liver with various replacing rates of humanhepatocytes and in chimeric mouse liver administered with rifampicin.

FIG. 11 shows test result on human albumin concentration in mice bloodfrom mice re-transplanted with human hepatocytes.

FIG. 12 is test result on the effect of retrorsine administering inuPA(+/+)/SCID mice.

FIG. 13 is test result on human albumin concentration in mice bloodadministered with an anti-mouse Fas antibody in human hepatocyteschimeric mice.

FIG. 14 illustrates a growth curve in culturing hepatocytes collectedfrom patients of various ages.

FIG. 15 is a phase-contrast microscopic image of cultured humanhepatocytes used as an antigen for preparing a monoclonal antibody.

FIG. 16 is an image of reactivity of hybridoma (K8233) culturesupernatant on human hepatocytes observed by immunofluorescencestaining.

FIG. 17 is a result analyzed by FACS of reactivity of hybridoma (No.23)culture supernatantat human hepatocytes surface immediately afterseparation.

FIG. 18 is a phase-contrast microscopic image in collection and cultureof a cell population reacted (R2) and non-reacted (R3) with hybridoma(No. 23) culture supernatant).

FIG. 19 is a result analyzed by FACS of reactivity of hybridoma (No. 23)culture supernatant at subcultured human hepatocytes surface.

FIG. 20 is a result analyzed by FACS of reactivity of hybridoma (K8216)culture supernatant at human, mouse and rat hepatocytes surfacesimmediately after isolation.

BEST MODE FOR CARRYING OUT THE INVENTION

The method for proliferating human hepatocytes of the 1st invention ischaracterized in that the transplanted human hepatocytes areproliferated in a mouse liver, wherein the human hepatocytes aretransplanted into the liver of an immunodeficient hepatopathy mouse andthen the recipient mouse is fed under such conditions as being protectedfrom the attack by a human complement produced by the human hepatocytes.

As described above, Mercer, et al. have reported that they prepared animmunodeficient hepatopathy mouse (uPA-Tg/SCID mouse) and the thawedhuman hepatocytes stored at a frozen state were transplanted therein,and as the results, about 50% of the liver was replaced by the humanhepatocytes (Nature Medicine 7:927-933, 2001). However, thehuman-hepatocytes-transplanted mouse prepared by Mercer, et al. couldnot achieve high replacement ratio by human hepatocytes.

The method of the 1st invention enables thehuman-hepatocytes-transplanted mouse to survive for not shorter than 50days by protecting the mouse from the attack by the human complementproduced by the transplanted human hepatocytes. Thus, extension ofviable time of a mouse for not shorter than 50 days made possible forthe mouse hepatocytes to be replaced by human hepatocytes by not lessthan 70%.

The 1st practical measure of protecting the recipient mouse from theattack by the human complement is administration of a “complementinhibitor” into a recipient mouse. An available complement inhibitorincludes, for example, nafamostat mesilate (Futhan®, ToriiPharmaceutical Co., Ltd.), gabexate mesilate (FOY®, Ono PharmaceuticalCo., Ltd.), comostat mesilate (Foipan®, Ono Pharmaceutical Co., Ltd.),and cobra venom factor (cobra toxin). By the way, these complementinhibitors are medical drugs usually used at cell transplantation ororgan transplantation, however, at ordinary operation oftransplantation, the complement inhibitors are used to protecttransplanted cells or organs from the attack by a complement produced bythe liver of the recipient. On the other hand, in the present invention,as the recipient mouse is considered not to produce its own complementdue to suffering from hepatopathy, a complement inhibitor is used toprotect the recipient mouse from the attack by a complement produced bytransplanted human hepatocytes.

The 2nd practical measure of protecting a recipient mouse from theattack by a human complement is the utilization of a progeniture mouse,as a recipient mouse, obtained by mating an immunodeficient hepatopathymouse and a human decay-accelerating factor (hDAF/CD55) transgenicmouse. It has been reported that, in hDAF/CD55 transgenic mouse, hDAFprotein expressed in high level on the surface of endothelial cells insuch an organ as a kidney, a heart, a lung and a liver, and therefore,said mouse had tolerance against a human complement (Murakami H,Takahagi Y, Yoshitatsu M, Miyagawa S, Fujimura T, Toyomura K, ShigehisaT, Shirakura R, and Kinoshita T.; Porcine MCP gene promoter directs highlevel expression of human DAF (CD55) in transgenic mice. Immunobiology,201:583-597, 1999/2000; van Denderen B J W, Pearse M J, Katerelos M,Nottle M B, Du Z-T, Aminian A, Adam W R, Shenoy-Scaria A, Lublin D M,Shinkel T A, and D'Apice A J F.; Expression of functionaldecay-accelerating factor (CD55) in transgenic mice protects againsthuman complement-mediated attack. Transplantation, 61:582-588, 1996).Therefore, the recipient mouse which is a progeniture animal between animmunodeficient hepatopathy mouse and a human decay-accelerating factor(hDAF/CD55) transgenic mouse has high tolerance against a humancomplement produced by transplanted human hepatocytes. The hDAF/CD55transgenic mouse may be prepared according to a well known method forproducing a transgenic mouse (for example, Proc. Natl. Acad. Sci. USA77:7380-7384, 1980) using a vector constructed according to theabove-described literatures (Immunobiology, 201:583-597, 1999/2000;Transplantation, 61:582-588, 1996) to be able to express hHDAF undercontrol of a promoter of, for example, a membrane cofactor protein (MCP)and H2K (b) (MHC class I) which have been expressed in wide variety oftissues and organs.

The “immunodeficient hepatopathy mouse” to be used for the method forproliferating human hepatocytes of the 1st invention is a “hepatopathymouse” in which native hepatocytes are dysfunction, and also an“immunodeficient mouse” which does not reject transplanted cells fromheterogeneous animals. Accordingly, the immunodeficient hepatopathymouse maybe prepared by giving both hepatopathy- andimmunodeficiency-inducing treatments to the same individual of mouse.The hepatopathy-inducing treatment includes, for example, a treatmentwith well known hepatopathy inducers (such as carbon tetrachloride,D-galactosamine, 2-acetylaminofluorene and pyrrolidine alkaloid) or asurgical hepatectomy, and the like. The immunodeficiency-inducingtreatment includes administration of an immune suppressor and surgicalthymectomy, and the like.

However, in the method of the 1st invention, a preferred embodiment isutilization of a progeniture mouse obtained by mating a mouse havinggenetic phenotype of hepatopathy and a genetically immunodeficient mouseas an immunodeficient hepatopathy mouse. For a genetically hepatopathymouse, above-described uPA-Tg mouse produced by Rhim, et al. (Science,263:1149 (1994)) may be exemplified. Alternatively, a transgenicimmunodeficient hepatopathy mouse may be prepared using a gene todevelop hepatopathy (for example, tissue-type plasminogen activatorgene, and the like) according to a well known transgenic method (Proc.Natl. Acad. Sci. USA 77:7380-7384, 1980). Also, a gene bearing liverfunction (for example, a fumarylacetoacetate hydrase gene, and the like)may be knocked out according to a well known gene-targeting method(Science 244:1288-1292, 1989) to obtain a genetically hepatopathy mouse.On the other hand, as a genetically immunodeficient mouse, well knownmouse, such as SCID mouse, NUDE mouse, RAG2 knockout mouse may be used.Hereinafter, a mouse obtained by the above procedures will be referredto as the “genetically immunodeficient hepatopathy mouse”.

As to the genetically immunodeficient hepatopathy mouse, it ispreferable to use a mouse having homozygote of a hepatopathy gene. Insuch a homozygous mouse, normal hepatocytes grow little, and therefore,proliferation of human hepatocytes is usually not inhibited by mousehepatocytes. Note, however, that when mating is carried out betweenhemizygous mice, provability of obtaining such a homozygous mouse willbe only ¼.

On the other hand, a genetically immunodeficient hepatopathy mousehaving a hemizygous hepatopathy gene (a “hemizygously immunodeficienthepatopathy mouse”) may be obtained with probability of ½ by matingbetween hemizygous mice, or by mating a hemizygous mouse and SCID mouse.This case enables to perform the 1st invention by reduced cost. However,in the hemizygously immunodeficient hepatopathy mouse, since one ofdiploid chromosomes is normal gene, normal hepatocytes beginproliferation with forming colony because of deletion of hepatopathygene, and at around 7 weeks after birth, normal mouse hepatocytes takeover the mouse liver. Therefore, in general, the mouse hemizygouslyimmunodeficient hepatopathy mouse is not suitable as a recipient mousefor proliferating human hepatocytes. And so, in the preferred embodimentof the 1st invention, before transplantation with human hepatocytes intothe hemizygously immunodeficient hepatopathy mouse, a specific inhibitorfor hepatocyte growth is administered to prevent from growth ofnormalized mouse hepatocytes to form colony. The hepatocyte growthinhibitor includes, for example, a kind of pyrrolidine alkaloids such asretrorsine, lasiocarpine, seneciphylline, monocrotaline andtrichodesmine.

In the method of the 1st invention, human hepatocytes to be used fortransplantation maybe isolated from a healthy human liver tissueaccording to a conventional method. A preferred embodiment, inparticular, of the method of the 1st invention, is use of proliferativehepatocytes having active proliferation potential in vivo. By the way,in the present invention, “proliferative human hepatocytes” means humanhepatocytes which form colonies as a single cell specie and proliferateso as to grow the colonies under in vitro culture condition. Such typeof growth may sometimes be referred to as “clonogenic proliferation”because the colony forming cells are composed of a single cell specie.Further, such cells can increase the cell population by subculture.

As an example of such proliferative human hepatocytes, the human smallhepatocytes invented by the present inventors of this application may beused. That is, the present inventors have found that the smallhepatocytes having high proliferation potential are contained in a rator human liver, and have applied for patents (JP-A-08-112092; JP No.3266766; U.S. Pat. No. 6,004,810, JP-A-10-179148; JP No. 3211941,JP-A-7-274951; JP No. 3157984, JP-A-9-313172; JP No. 3014322). Relatingarticles thereof have been published (Tateno, C. and Yoshizato, K.;Growth and differentiation in culture of clonogenic hepatocytes thatexpress both phenotypes of hepatocytes and biliary epithelial cells. Am.J. Phathol. 149: 1593-1605, 1996; Hino, H. Tateno, C. Sato, H. Yamasaki,C. Katayama, S. Kohashi, T. Aratani, A. Asahara, T. Dohi, K. andYoshizato, K.; A long-term culture of human hepatocytes which show ahigh growth potential and express their differentiated phenotypes.Biochem. and Biophys. Res. Commun. 256: 184-191, 1999; Tateno, C.Takai-Kajihara, K.; Yamasaki, C. Sato, H. and Yoshizato, K.;Heterogeneity of growth potential of adult rat hepatocytes in vitro.Hepatology 31: 65-74, 2000; and Katayama, S. Tateno, C. Asahara, T. andYoshizato, K.; Size-dependent in vivo growth potential of adult rathepatocytes. Am. J. Pathol. 158: 97-105, 2001). Said human smallhepatocytes grow rapidly in a recipient body, and can form a humanhepatocyte population having normal liver function in short time due tosuperior proliferation potency.

Such small hepatocytes may be collected by the method usingcentrifugation described in the above prior inventions, or a methodusing a cell fractionator such as an elutoriator and FACS. Further,hepatocytes proliferating with forming colony can also be collectedusing a monoclonal antibody specifically recognizing them. The source ofsmall hepatocytes may be in vitro proliferated human hepatocytes, storedhepatocytes in a frozen state, immortalized hepatocytes by introductionof such as a telomerase gene, and a mixture of these hepatocytes andnon-parenchymal cells.

Another example of proliferative human hepatocytes includes humanhepatocytes recognized by the monoclonal antibody (Example 6) specificfor proliferative human hepatocytes. Such proliferative humanhepatocytes recognized by a specific monoclonal antibody may be obtainedin high purity by a well known method such as EIA, RIA and fluorescentantibody technique, depending on labels of the antibody. Concretely,separation of the proliferative human hepatocytes can be performedwhereby a monoclonal antibody labeled with enzyme, radioisotope,magnetic beads or fluorescent pigment is brought into contact with ahuman hepatocytes population and hepatocytes indicating labeled signalare isolated. The enzyme used for labeling is not specifically limited,as long as it can satisfy conditions such as having large turnovernumber, exhibiting stability in a state of binding to an antibody andspecifically coloring substrate; and includes enzyme generally used inEIA, such as peroxidase, β-galactosidase, alkaline phosphatase, glucoseoxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase andmalate dehydrogenase. An enzyme inhibitor and a coenzyme can also beused. Binding between these enzymes and the antibody can be achieved bya known method using a crosslinking agent such as a maleimide compound.With regard to substrate, known substance can be used depending on typesof enzyme used. For example, when peroxidase is used as the enzyme,3,3′,5,5′-tetramethylbenzidine can be used, and when alkalinephosphatase is used as the enzyme, such as p-nitrophenol can be used.With regard to the radioisotope, conventionally used radioisotope in RIAsuch as ¹²⁵I and ³H can be used. With regard to the fluorescencepigment, those conventionally used for fluorescence antibody techniquesuch as fluorescence isothiocyanate (FITC) and tetramethylrhodamineisothiocyanate (TRITC) can be used. When a enzyme is used, detection oflabeled signal can be performed by adding substrate, which developscolor by decomposition due to enzyme action, followed by assaying enzymeactivity by measuring the decomposed amount of the substrate byphotometry, converting to an amount of bound antibody, and calculatingthe antibody amount by being compared with the standard value. When aradioisotope is used, radiation quantity emitted by the radioisotope ismeasured by using a scintillation counter, and the like. When afluorescence pigment is used, fluorescence yield is measured byapparatus combined with a fluorescence microscope.

As described above, human hepatocytes, especially proliferative humanhepatocytes, can be transplanted into an immunodeficient hepatopathymouse via a mouse spleen, as described in the Example. Also, directtransplantation via a portal vein may be possible. Number of humanhepatocytes to be transplanted may be approximately in the range from 1to 10,000.

Further, in the preferred embodiment of the method of the 1st invention,an anti-mouse Fas antibody is administered into a mouse transplantedwith human hepatocytes. An anti-mouse Fas antibody induces apoptosis byreacting with the Fas antigen on mouse hepatocytes. Administration ofthe anti-mouse Fas antibody into the mouse transplanted with humanhepatocytes, causes mouse hepatocytes apoptosis, and leads them to die.Since this antibody does not affect human hepatocytes, only transplantedhuman hepatocytes may proliferate selectively. By this method,replacement ratio of human hepatocytes in mouse liver may further beincreased.

A method for proliferating human hepatocytes in large scale in the 2ndinvention will be explained next.

The method of the 2nd invention is characterized by carrying out thefollowing steps (1) to (3).

-   -   Step (1): Human hepatocytes are proliferated in a mouse liver        similarly as in the method of the 1st invention.    -   Step (2): Human hepatocytes proliferated in a mouse liver are        isolated.    -   Step (3): The isolated human hepatocytes are transplanted        respectively into immunodeficient hepatopathy mice, and the        human hepatocytes are proliferated similarly as in the method of        the 1st invention.

In addition, in the method of the 2nd invention, the above-describedsteps (2) and (3) are repeated at least once. That is, in the step (1),human hepatocytes transplanted into a mouse will increase the cellnumber up to 100 times in the mouse liver. Therefore, if all theproliferated human hepatocytes are recovered in the step (2), inprinciple, 100 mice may be transplanted with human hepatocytes at thestep (3), and finally, human hepatocytes of 100×100 times initiallytransplanted human hepatocytes may be obtained. Further, by repeatingthe steps (2) and (3) twice, human hepatocytes of 100×100×100 timesinitially transplanted human hepatocytes may be recovered.

In the method of the 2nd invention, the step (1) and/or the step (3) maybe conducted by the same procedures as in the above-described method ofthe 1st invention and a preferred embodiment thereof. The term above“and/or” means that one requirement in the method of the above-described1st invention may be performed either in the step (1) only, or in thestep (3) only, or in both steps (1) and (3). For example, in the step(1), proliferative human hepatocytes recognized by the above-describedmonoclonal antibody (the 8th invention) are transplanted into a mouse,and in the step (3), all of the recovered human hepatocytes may be usedfor transplantation. Each requirement in the above-described 1stinvention may be selected appropriately depending on individual mouse inwhich human hepatocytes are proliferated, applications of the humanhepatocytes recovered from a mouse, and the like.

The step (2) of the 2nd invention is a process where the proliferatedhuman hepatocytes in the step (1) [the just before step, in the casewhen the steps (2) and (3) are repeated] are isolated from a mouseliver. As to the separation method, various methods (for example, acollagenase perfusion method, and the like) may be applied, however,application of at least one of the following methods (a) and (b) ispreferable.

Method (a):

By treatment of a liver tissue separated from a mouse liver withcollagenase, substantially only human hepatocytes are recovered. Ascytotoxicity of collagenase is higher for mouse hepatocytes than forhuman hepatocytes, mouse hepatocytes may be damaged dominantly bycontrolling digestion period with collagenase, and only humanhepatocytes may be collected. Treatment period by collagenase depends onratio between human hepatocytes and mouse hepatocytes, however, forexample, if content of human hepatocytes is not lower than 70%,treatment period may be from 8 to 20 minutes.

Method (b):

By isolation of cells recognized by a monoclonal antibody whichspecifically recognizes human hepatocytes but not recognizes non-humanhepatocytes (the 8th invention), highly pure human hepatocytes arerecovered. Such a monoclonal antibody may be exemplified by themonoclonal antibody produced by Mouse-Mouse hybridoma K8216 (FERMP-18751).

The 3rd invention of the present application is a “chimeric mouse”carrying human hepatocytes in the liver, wherein human hepatocytes areproliferated by the methods of the above-described 1st invention or 2ndinvention. The chimeric mouse is preferably one lived not shorter than50 days, more preferably not shorter than 70 days from transplantationof human hepatocytes. In addition, the chimeric mouse is preferably one,having replacement ratio of mouse liver by human hepatocytes is notlower than 70%, more preferably not lower than 90%, and particularlypreferably not lower than 98%. Further, the chimeric mouse is one havinghuman cytochrome P450 activity. That is, there are dozens of isozymes inhuman-type P450, such as CYP1A1, 1A2, 2C9, 2C19, 2D6 and 3A4, however,the chimeric mouse of the present invention is characterized byexpressing these human-type P450 substantially in the same level ashuman hepatocytes. The cytochrome P450 is protein taking part inmetabolism of ecdemic chemical substances in a liver, and therefore, amouse liver expressing human-type P450 may metabolite chemicalsubstances or the like substantially in similar manner as a human liver.Accordingly, the chimeric mouse of the 3rd invention enables a methodfor testing, for example, influence of efficacy or toxicity of medicaldrugs on human hepatocytes (the 9th invention). By the way, the priorinvention of the present inventors (JP-A-2002-45087: chimeric animals)may be referred to on application of this kind of chimeric mouse. Inaddition, a population of a chimeric mouse carrying in the liver humanhepatocytes proliferated in large scale by the method of the 2ndinvention, is a population of chimeric mouse each carrying the samehuman-derived hepatocytes, and thus, enables to conduct a large-scaletest under a substantially uniform condition.

The 4th invention of the present application is the method for isolatinghuman hepatocytes from the chimeric mouse of the above-described 3rdinvention. This method may be carried out similarly as in the step (2)of the above-described 2nd invention.

The 5th invention of the present application is human hepatocytesisolated from the liver of the chimeric mouse by the method for theabove-described 4th invention. These human hepatocytes havesubstantially the same liver functions as normal hepatocytes in a humanliver tissue by virtue of proliferating in an individual mouse.Accordingly, by application of the human hepatocytes of the 5thinvention, a human hepatocytes kit (the 6th invention) which may be usedfor a drug metabolism test and a safety test, or a hybrid-typeartificial liver (the 7th invention) may be provided. Further, using amodule (a module filled with human hepatocytes) to be used for ahybrid-type artificial liver, valuable substances produced by humanhepatocytes may be recovered.

As to a hepatocytes kit, various types of kits depending on cell speciesand applications are well known. Therefore, the cellular kit of the 6thinvention may easily be prepared by those skilled in the art by adoptingthe human hepatocytes of the 5th invention and compositions of wellknown cellular kits. Also, as the compositions of modules andhybrid-type artificial organs are well known, it is easy for thoseskilled in the art to prepare the artificial liver of the 7th invention.

In addition, the 8th invention of the present application provides amonoclonal antibody which specifically recognizes human hepatocytes anddoes not recognize non-human hepatocytes.

This monoclonal antibody may be prepared according to well known methodfor preparation of a monoclonal antibody (“Monoclonal Antiobody”Nagamune H?. and Terada H.; Hirokawashoten, 1990; “Monoclonal Antiobody”James W. Goding, third edition, Academic Press, 1996), for example,according to the following procedures.

1. Preparation of Hybridoma Cells

Mammalians are immunized by using an immunogen containing subculturedhuman normal hepatocytes, and if necessary, animals are sufficientlysensitized by immunizing properly and additionally. Antibody producingcells (lymphocytes or spleen cells) are then collected from the animalsto obtain hybrid cells between these antibody producing cells andmyeloma cell lines. Cells which produce an objective monoclonal antibodyare selected from these hybrid cell lines and cultured to obtainhybridoma cells. Each process is explained in detail below.

a) Preparation of an Immunogen

Human hepatocytes separated from normal liver tissues using collagenaseare subcultured to prepare an immunogen. The human hepatocytes separatedfrom human liver tissues not older than 15 years old, which are able tosubculture for not less than 4 passages, preferably not less than 6passages, are preferably used.

b) Immunization of Animals

Animals to be immunized can be mammals used for known hybridomapreparation and include typically, for example, mice, rats, goats,sheep, bovines and equines. Mice or rats are preferable animals forimmunization from the standpoint of easy availability of myeloma cellsto be fused with separated antibody producing cells. There is nospecific limitation on strains of mice and rats actually used, andstrains of mice such as A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BR,C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, RIII, SJL, SWR, WB and 129,and strains of rats such as Low, Lewis, Sprague, Daweley, ACI, BN andFisher can be used. Considering compatibility with myeloma cells, whichwill be explained hereinbelow, BALB/c strain for mouse and Low strainfor rat are particularly preferable for animals to be immunized. Ages ofthese mice and rats for immunization are preferably 5-12 weeks old.

Immunization of animals can be performed by administering subculturedhuman hepatocytes as the immunogen, about 10⁴-10⁸ cells,intracutaneously or intraperitoneally into the animals. Administeringschedule of the immunogen depends on types of animals to be immunized,individual differences, etc., however generally frequency ofadministering the immunogen is 1-6 times and an administering intervalis 1-2 weeks in case of multiple administrations.

c) Cell Fusion

Spleen cells or lymphocytes containing antibody producing cells areaseptically collected from the animals to be immunized after 1-5 daysfrom the final immunization date on the above administering schedule.The separation of the antibody producing cells from spleen cells orlymphocytes can be performed according to a known method.

The antibody producing cells and the myeloma cells are then fused. Themyeloma cells used are not specifically limited and can be selectedappropriately from known cell lines for use. However, consideringconvenience in selection of hybridoma from the fused cells, use of HGPRT(Hypoxanthine-guanine phosphoribosyltransferase) deficient strain,wherein selection procedure thereof has been established, is preferable.Namely, those are X63-Ag8(X63), NS1-Ag4/1(NS-1), P3X63-Ag8.U1(P3U1),X63-Ag8.653(X63.653), SP2/0-Ag14(SP2/0), MPC11-45.6TG1.7(45.6TG), FO,S149/5XXO, BU.1, etc. derived from mice; 210.RSY3.Ag.1.2.3(Y3), etc.derived from rats; and U266AR(SKO-007), GM1500·GTG-A12(GM1500), UC729-6,LICR-LOW-HMy2(HMy2), 8226AR/NIP4-1(NP41), etc. derived from human.

Fusion of the antibody producing cells and the myeloma cells can beperformed as appropriate under conditions without extremely reducingcell viability according to a known method. Such a method includes, forexample, a chemical method for admixing the antibody producing cells andthe myeloma cells in a high concentration solution of a polymer such aspolyethylene glycol, and a physical method utilizing electricstimulation.

Selection of fused cells and non-fused cells are preferably performed,for example, by a known HAT (hypoxanthine-aminopterin-thymidine)selection method. This method is effective for obtaining fused cells byusing the myeloma cells of HGPRT deficient strain which can not viablein the presence of aminopterin. Namely, fused cells with resistant toaminopterin can selectively be left and proliferated by culturingnon-fused cells and fused cells in HAT medium.

d) Screening of Hybridoma

Screening of hybridoma cells producing an objective monoclonal antibodycan be performed by known methods such as enzyme immunoassay (EIA),radio immunoassay (RIA) and fluorescent antibody technique. Thehybridoma cells producing a monoclonal antibody specifically whichrecognizes human hepatocytes but not recognizes non-human hepatocytescan be obtained by such a screening method.

The screened hybridoma cells are cloned by known methods such as amethylcellulose method, a soft agarose method and a limiting dilutionmethod and are used for antibody production.

The hybridoma cells obtained by methods explained hereinabove can bestored under a freezing state in liquid nitrogen or a freezer at nothigher than −80° C. The present application provides Mouse-Mousehybridoma K8216 (FERM BP-8333), as a typical example of such a hybridomacell.

2. Obtaining a Monoclonal Antibody and Purification Thereof

A monoclonal antibody, which is specifically bound to human hepatocytesand never recognizes any non-human hepatocytes, can be obtained byculturing the hybridoma cells prepared in the above-described 1 with aknown method.

A cultivation may be performed, for example, in medium having the samecomposition used in the above cloning method; or to obtain themonoclonal antibody in large scale, may be performed by a method whereinthe hybridoma cells were intraperitoneally injected in mouse and themonoclonal antibody is collected from ascites.

Thus obtained monoclonal antibody can be purified, for example, bymethods such as ammonium sulfate fractionation, gel filtration, ionexchange chromatography and affinity chromatography.

The present application provides the monoclonal antibody produced by theabove-described hybridoma cell, Mouse-Mouse hybridoma K8216 (FERMBP-8333) as a typical example of the monoclonal antibody.

This monoclonal antibody of the 8th invention may be used for separatingonly human hepatocytes from a mouse liver in the above-described methodsof the 2nd and the 4th inventions. Besides, since the monoclonalantibody of the 8th invention does not recognize non-human hepatocytes,said antibody may be used for isolating and purifying human hepatocyteswhen human hepatocytes are proliferated in a body of a non-human animalother than a mouse, or when non-human hepatocytes and human hepatocytesare mix cultured.

In addition, “a monoclonal antibody which specifically recognizesproliferative human hepatocytes” used in the present invention may alsobe prepared by the same method as described above (see Example 6),except for selecting a hybridoma producing an objective antibody, in ascreening process. The present application provides the monoclonalantibody produced by Mouse-Mouse hybridoma K8223 (FERM BP-8334) as atypical example of such a monoclonal antibody.

EXAMPLES

Some tests exemplifying the present invention are described below;however, the scope of the present invention should not be limitedthereto.

Example 1 Preparation of a Chimeric Mouse Carrying Proliferated HumanHepatocytes

A chimeric mouse carrying proliferated human hepatocytes in the liverwas prepared by transplanting human hepatocytes into the liver of animmunodeficient hepatopathy mouse. Materials and methods for thepreparation and the results of the relevant testing were as follows:

1. Materials

-   (a) an albumin-urokinase plasminogen activator transgenic mouse    (uPA-Tg): B6SJL-TgN (Alb1Plau) 144Bri-   (b) SCID mouse (Scid): C.B-17/Icr Scidjcl-   (c) nafamostat mesilate-   (d) retrorsine-   (e) human hepatocytes

The mouse (a) and the mouse (b) were purchased from The JacksonLaboratory and CLEA Japan. Co., respectively. The medical agent (c)(brand name: Futhan®) and (d) were purchased from Torii Pharmaceuticaland SIGMA Co., respectively.

The cells (e) were prepared as described below. Before operatinghepatectomy, agreement to the informed consent by patient was completed.A normal liver tissue obtained from the removed liver was perfused withUW solution, and then transported at 4° C. The normal liver tissue wastreated with collagenase to obtain a dispersed solution of a liver,followed by low-speed centrifugation (50×g, for 2 minutes) to separateprecipitate and supernatant, washing the cells contained in each part(supernatant: small hepatocytes, precipitate: parenchymal hepatocytes)with culture medium and respectively counting cell number. When cellswere stored in a frozen state, freeze-preservation liquid (from KURABOCo.) was added and then frozen using a programmed freezer. The frozenhepatocytes from the precipitate and the supernatant, stored in liquidnitrogen, were thawed at 37° C. in a constant temperature water bath,and cell number was counted. Cell concentration was adjusted to 4×10⁷cells/ml using medium just before transplantation.

2 Methods and Results

2.1 Preparation of a Mouse Suffering from Immunodeficient Hepatopathy

The uPA-Tg mouse (hemizygote, ±) and the SCID mouse (homozygote, ±) weremated, and Tg(±)/SCID(±) mouse having phenotype of both parents wasobtained in 35.2% probability. Identification of Tg(±) and Tg(−/−) wasperformed by a genome PCR method using a primer with a specific sequencefor Tg gene. Also, identification of SCID(±) and SCID(−/−) was performedby PCR-RFLP method.

Thus obtained Tg(±)/SCID(±) mouse was then backcrossed with theSCID(+/+) mouse, to obtain Tg(±)/SCID(+/+) mouse. As the results, Tg(±)emerged in 37.9% probability and SCID(+/+) emerged in 52.8% probability.The objective Tg(±)/SCID(+/+) and Tg(+/+)/SCID(+/+) mice were obtainedby mating Tg(±) /SCID(+/+) mice themselves.

Identification of homozygote and heterozygote of uPA genes was performedby the following method.

From 5 mm of a tail cut off from 8 to 10 days old mouse, genomic DNA wasextracted using the Qiagen DNeasy Tissue Kit. Concentration and purityof the extracted DNA were measured using a spectrophotometer. To amixture of 25 μl of master mix, 1 μl of primer F, 1 μl of primer R and 1μl of Taqman probe, DNA and distilled water were added, and the totalvolume was adjusted to 50 μl to be subjected to quantitative PCR (ABI7700, Sequencer Detector, PE Applied Biosystems).

The primers and Taqman probe were designed for directing to humangrowth-hormone-encoding sequence incorporated in a vector for uPA genetransfer as shown below. Primer F: gtcttggctcgctgcaatc (SEQ ID No:1)Primer R: cgggagactgaggcaggag (SEQ ID No:2) Taqman probe:ccgcctcctgggttcaagcga (SEQ ID No:3)

As a control, the following primer and Taqman probe directing to mouseG3PDH-encoding sequence were used. Primer F: ggatgcagggatgatgttc (SEQ IDNo:4) Primer R: tgcaccaccaactgcttag (SEQ ID No:5) Taqman probe:cagaagactgtggatggccctc (SEQ ID No:6)

The PCR was carried out under the condition that after denaturation at95° C. for 10 minutes, 50 cycles of denaturation at 95° C. for 15seconds and elongation at 60° C. for 1 minute were repeated. From therelative value obtained by dividing the amount of amplified fragment ofhuman growth-hormone-encoding sequence by the amount of amplifiedfragment of mouse G3PDH-encoding sequence as an internal control, theamount of introduced gene in genomic DNA is compared. A standard curvewas obtained from sequential dilution of heterozygous or homozygousgenome of a sample mouse, and the relative value of a mouse used for thestandard curve was defined as 1. If the standard mouse is heterozygousone, a heterozygous mouse shows close value to 1 and a homozygous mouseshows close value to 2. However, if the standard mouse is homozygousone, a homozygous mouse shows close value to 1 and a heterozygous mouseshows close value to 0.5. From naked eye observation at anatomy, thehitting ratio is supposed to be about 90%.

2.2 Transplantation of Human Hepatocytes into the uPA-Tg/SCID Mouse

The Tg(+/+)/SCID(+/+) mouse, from 14 to 48 days old, was anesthetized byether and incised about 5 mm at the flank, and then, from 10 to 12.5 μlof cell suspension with concentration from 4 to 8 x 10⁷cells/ml (from 4to 10×10⁵ cells in total) was inoculated from a spleen head, and thenstanched. As a hemostatic agent, 40 μl of an e-aminocaproic acid (SIGMA)solution (0.02 g/ml) was administered into the abdominal cavity, andthen, after the spleen was returned to the abdominal cavity, the flankwas sewed up. Concentration of uPA in mouse blood is high, because theuPA produced by hepatocytes of a Tg mouse is secreted extracellularly.The uPA has activity of catalyzing proteolysis and activation ofplasminogen to plasmin, and decomposing fibrin clot. Hence, it has beensaid that a number of mice will die with bleeding in an intestinal ductand an abdominal cavity within 4 days after birth. To avoid death bybleeding on surgery, e-aminocaproic acid was administrated, which hashemostatic effects by inhibiting actions of a plasminogen activator anda plasmin.

The SCID/C.B-17 mouse used for mating is known to have neither T-cellsnor B-cells but have NK cells. Therefore, to protect the transplantedhuman hepatocytes from the attack by NK cells, an asialo GM1 antibodywhich inhibits NK activity was inoculated into an abdominal cavity theday before and the day after transplantation. The progeniture of mousewas weaned at 4 weeks after birth, and each male and female was fed in aseparate cage after 5 weeks.

2.3 Monitoring of Human Albumin in Mouse Blood

From 1 week after transplantation of human hepatocytes, 10 μl bloodsamples were taken from the tail of a mouse once or twice per a week,and concentration of human albumin was measured using Quantitative ELISAimmunoassay (Bethyl Laboratories Inc.).

Increased concentration of human albumin in blood was observed in 11mice out of 19 Tg(+/+)/SCID mice which had been transplanted with humanhepatocytes (FIG. 1). The highest value was over 8 mg/ml correspondingto 62% of total albumin in mouse blood.

Further, human albumin concentrations in blood were compared betweenmice transplanted with cells in supernatant (small hepatocytes) and micetransplanted with cells in precipitate (parenchymal hepatocytes)separated by low speed centrifugation of human hepatocytes. Higherincrease in human albumin was observed in the mice transplanted with thesmall hepatocytes than those with the parenchymal hepatocytes (FIG. 2).

2.4 A Human Complement Produced by Human Hepatocytes and Administrationof a Complement Inhibitor

Increase in concentration of human albumin in blood over 3 mg/ml wasobserved in mice which rapidly worsen mouse condition and led to death(FIG. 1). In the dead mice, bleeding was observed in lungs thereof, andaffects of a complement produced by human hepatocytes were envisaged.Also, human hepatocytes in the mouse liver tissue were stainedimmunologically using an anti-human complement C3 antibody, and presenceof a complement C3 in human hepatocytes was confirmed. Therefore, 200 μlof a complement inhibitor (Futhan®) with concentration of 2 mg/ml wasadministered into the mouse with increased human albumin concentration.Administration frequency was started from once per 2 days. Change inbody weight was checked every day, and administration frequency or dosewas increased when decrease in body weight was observed (FIG. 3). As theresults, the mice with human albumin concentration in blood over 2 mg/mlreceived the complement inhibitor (Futhan®) every 2 days, and those over4 mg/ml received every day, and further, those over 6 mg/ml receivedtwice a day (FIG. 3). By this treatment, mouse could live for longperiod even when human albumin concentration in blood exceeded 3 mg/ml,and the highest detectable level of human albumin in blood was 8 mg/ml(FIG. 1).

Further, concentration of the human complement (hC3a) in blood ofchimeric mice administered or non-administered with the complementinhibitor (Futhan®) was measured by ELISA method. In consequence, it wasconfirmed that hC3a concentration was lower in chimeric miceadministered with the complement inhibitor compared withnon-administered mice (FIG. 4).

2.5 Administration of Vitamin C and Serum of SCID Mouse

Possibility of vitamin C deficiency was considered in mouse having aliver replaced with human hepatocytes because human hepatocytes can notsynthesize vitamin C. Therefore, ascorbic acid 2-phosphate dissolved indrinking water in 1 mg/ml was given to the mouse transplanted with humanhepatocytes (by referring to “A method for feeding a rat with deficiencyof ascorbic acid synthesis”; CLEA Japan Co.).

Further, a human hepatocytes-transplanted Tg(+/+)/SCID mouse showedslower increase in body weight and worse hair appearance, compared witha Tg(±)/SCID mouse. As it was considered that proteins, enzymes and thelike synthesized by human hepatocytes in a mouse liver were insufficientfor growth and maintenance of mouse body, 50 μl of SCID mouse serum wasadministered subcutaneously once per a week. Increase in body weight ofTg(+/+)/SCID administered with SCID mouse serum was higher compared withnon-received mice.

2.6 Gross and Histopathological Examination of a Mouse Liver

Blood was collected from a mouse under anesthesia with ether to measureserum albumin and GPT value. A liver and a spleen were removed tomeasure weight, followed by photographing, sampling for a frozen sectionblock and a paraffin section block and extracting a microsomal fractionfrom the rest of the samples. Increase in human albumin concentration,along with increase in serum albumin and decrease in GPT value wereobserved (FIG. 5).

In mice with high human albumin value, whole liver looked pinkish, butin some mice, remaining white spot, which is a distinctive phenotype ofTg(+/+)/SCID, was observed. Also, there observed red color part in theliver, which part was considered as normalized mouse hepatocytes causedby deletion of introduced uPA gene.

Frozen sections of each lobe of the liver were prepared, followed byreacting with the human specific cytokeratin 8/18 antibody (from ICNPharmaceuticals, Inc., Ohio) and staining by peroxidase labeled DAKOEnvision⁺™ (from DAKO Corporation, CA). With respect to each lobe of theliver, ratio of cytokeratin 8/18 positive areas in the total sectionarea was calculated (FIG. 6, FIG. 7). In mice with high human albuminvalue, a percentage of the area of cytokeratin 8/18 positive hepatocyteswas high. A mouse having over 80% of positive cells was also observed.

2.7 Analysis of P450 Isozymes in Microsome

Expression of P450 isozymes in a microsomal fraction collected from amouse was detected by western-blotting analysis using antibodies whichspecifically recognize the P450 isozyme (from Daiichi Pure ChemicalsCo., Ltd.). Expression of CYP2C9, a human specific P450 isozymes, wasobserved in mice with high human albumin value (FIG. 8).

Further, to check activity of CYP2C9 in the microsomal fraction sampledfrom a mouse, diclofenac 4-hydroxylation activity was measured by addingdiclofenac to the microsome. In consequence, diclofenac 4-hydroxylationactivity was little detected in the uPA(−/−)/SCID mouse, and inductionof activity by administration of a complement inhibitor was not observedalso. On the other hand, in human-hepatocytes-transplanted chimericmice, higher diclofenac 4-hydroxylation activity was observed withincrease in replacement ratio of human cells. In a chimeric mouse with89% of replacement ratio, diclofenac 4-hydroxylation activity wasconfirmed higher than that of donor (human) microsome (FIG. 9).

2.8 Expression of niRNAs of Respective P450 Isozymes in a Liver of aChimeric Mouse, and Induction by Rifampicin

Total RNA was extracted from a chimeric mouse, and corresponding cDNAwas synthesized by reverse transcription reaction. Primers for 6 typesof cDNA corresponding to each P450 is ozyme(CYP1A1, 1A2, 2C9, 2C19, 2D6and 3A4)-encoding gene were synthesized, to measure expression of mRNAcorresponding to each isozyme, using PRISM 7700 Sequence Detector (fromApplied Biosystems Inc.). Consequently, a control mouse with replacementratio of 0% expressed almost zero human-type P450, however, all ofhuman-type P450 isozymes were expressed in a chimeric mouse liver, andthe expression pattern was closely resembled to that of a donor (human)(FIG. 10).

Further, 3 chimeric mice were administered intraperitoneally withRifampicin (50 μg/kg body weight) for 4 days, followed by taking out theliver at 5 days and assaying the expression of 6 human-type P450similarly as described above. It is well known that Rifampicin inducesexpression of only human CYP3A4. As the result, the expression level ofhuman-type CYP3A4 in the liver of the Rifampicin-administered chimericmice were about 5.7 times higher compared with that of non-administeredmice (FIG. 10).

Example 2 Separation of Human Hepatocytes from a Chimeric Mouse, andPurification of Human Hepatocytes with a Monoclonal Antibody

From the chimeric mouse prepared in Example 1, hepatocytes wereseparated by a collagenase perfusion method. Concentration ofcollagenase was 0.05%, and treating period was 9 minutes. It wasconceivable that hepatocytes was a mixture of human hepatocytes andmouse hepatocytes, however, most of hepatocytes separated (about 80%)were human hepatocytes because cytotoxicity of collagenase is higher formouse hepatocytes than for human hepatocytes.

Further, to increase purity of human hepatocytes, only human hepatocyteswere isolated using a mouse monoclonal antibody (the monoclonal antibodyproduced by the hybridoma K8216 in Example 6) which does not recognizemouse hepatocytes but specifically recognizes surface of humanhepatocytes. The hepatocytes separated by collagenase perfusion, whichcontains about 80% of human hepatocytes, were reacted with theabove-described antibody, followed by reaction with an FITC-labeledmouse IgG antibody and separation of cells reacted with the FITC-labeledmouse IgG antibody, using fluorescence activated cell sorter (FACS). Asthe result, purity of human hepatocytes went up to not lower than 95%.

Example 3 Re-Transplantation of Human Hepatocytes Isolated from aChimeric Mouse into a uPA-Tg/SCID Mouse

The hepatocytes isolated in Example 2 were transplanted into anotheruPA-Tg(+/+)/SCID mouse, and the transplanted mouse was fed similarly asin Example 1. As the result, increase in human albumin level in mouseblood was observed (FIG. 11).

Example 4 Growth Inhibition of Normalized Mouse Hepatocytes Caused byDeletion of Introduced uPA Gene

Retrorsine was inoculated intraperitoneally in the amount of 30 or 60mg/kg into a uPA-Tg(±)/SCID mouse and a uPA-Tg(+/+)/SCID mouse. As theresult, number of colonies of normalized hepatocytes caused by deletionof introduced uPA gene (uPA-Tg(−/−)/SCID) was reduced in theuPA-Tg(±)/SCID mouse (FIG. 12).

Then, the uPA-Tg(±)/SCID and the uPA-Tg(+/+)/SCID mice, which wereinoculated with 60 mg/kg of retrorsine intraperitoneally, weretransplanted with human hepatocytes similarly as in Example 1 to preparechimeric mice. As the result, human albumin concentration in the bloodof the chimeric uPA-Tg(±)/SCID mouse was also increased to the samelevel as that of the chimeric uPA-Tg(+/+)/SCID mouse.

Example 5 Administration of an Anti-Mouse Fas Antibody into a HumanHepatocytes Chimeric Mouse

As to one chimeric mouse prepared in Example 1, at 100 days aftertransplantation of human hepatocytes, an anti-mouse Fas antibody whichleads mouse hepatocytes to apoptosis by reaction with a Fas antigenthereof was inoculated (0.2 μg/g body weight) into the abdominal cavityof said chimeric mouse twice at the interval of 1 week.

As the results shown in FIG. 13, in this chimeric mouse, concentrationof human albumin in blood thereof indicates a declining tendency afterabout 80 days from transplantation of human hepatocytes, however,concentration of those was significantly increased by administration ofthe anti-mouse Fas antibody.

In consequence, in the method of the present invention, it was confirmedthat administration of the anti-mouse Fas antibody into a chimeric mousejust after transplantation of human hepatocytes was effective toproliferation and/or activation of human hepatocytes.

Example 6 Preparation of a Monoclonal Antibody Which SpecificallyRecognizes Human Proliferative Hepatocytes

1. Culture of Human Hepatocytes

A dispersed cell suspension was obtained from a human liver tissue by acollagenase perfusion method. The dispersed cell suspension wassubjected to low-speed centrifugation (50×g, for 2 minutes), and thesedimentary fraction was co-cultured with mitomycin C-treated Swiss 3T3cells using Dulbecco's modified Eagle's medium (DMEM) added with fetalcalf serum, human serum, EGF, nicotinamide and activity-persistent typevitamin C. Swiss 3T3 cells were added every ten days. Human hepatocytescolonies were observed after about 7 days of culture. Proliferatedhepatocytesin confluent were subcultured by using EDTA/Trypsin.Hepatocytes of children could be subcultured up to 6-9 passages,however, hepatocytes of patients, not younger than 60 years old, couldonly be subcultured until 3-4 passages (FIG. 14). The hepatocytes of thechild (12 years old) exhibiting the highest proliferation ability wereused as an antigen (FIG. 15).

2. Immunization of Animals

The hepatocytes of the child (12 years old) at 3-5 passages by theabove-described method were proliferated on culture dishes. Afterconfluent-proliferated cells (about 1×10⁷ cells) were washed with PBS (aphosphate buffer salt solution), PBS was removed and the cells werescraped with a cell scraper to suspend in about 1 ml PBS. Thus obtainedsuspension was administered intraperitoneally in Balb/c mice, 6 weeksold. Immunization was further performed by a similar method after 20days or 30 days.

3. Cell Fusion

After twice immunization, increased antibody titer was observed. After72 hours of the third immunization (boost), a spleen was extracted froman immunized animal to collect spleen cells. These spleen cells andmouse myeloma cells (cell line name: NS-1) were fused, plated into 372wells of 96-well plates and cultured.

4. Screening of Hybridoma

Primary Screening (ELISA, Tissue Staining)

Reactivity of culture supernatant of the fused cells thus obtained to anantigen was assayed by ELISA. Assay was performed by the followingmethod. The subcultured hepatocytes used as the antigen were plated onthe 96-well plate, followed by washing with PBS after cultivation,drying and storing at −80° C., to which the culture supernatant wasreacted. An enzyme labeled anti-mouse IgG antibody or anti-mouse IgMantibody was then reacted thereto, followed by developing color byadding substrate to measure absorbance. As the results, mean absorbanceof the 372 fused cell samples was 0.149 (SD: 0.099), and samples withabsorbance of not lower than 0.20 (81 samples, about 20%) were definedas positive samples. Since color development was also confirmed in nakedeye observation in samples with absorbance of not lower than 0.15,samples with the absorbance of from 0.15 to 0.20 (46 samples) weretreated with tissue staining to confirm reactivity. Among them, only 13samples showing interesting staining pattern were selected as positivesamples. Selected 94 positive samples were further cultured in largescale, and culture supernatant was recovered and cells were stored in afrozen state.

5. Secondary Screening (ELISA, Tissue Staining)

From 94 samples selected by the primary screening, 88 positive sampleswere selected by measuring reactivity against an antigen in culturesupernatant after the large scale cultivation, by ELISA similarly as inthe primary screening. Reactivity in tissue of these samples was studiedby tissue staining. Samples containing hybridoma, which specificallyreacts with cell membrane of hepatocytes and hepatocytes in portalregion, or culture supernatant of the clone obtained by cloningtherefrom, were studied on reactivity against human hepatocytesimmediately after separation.

Supernatant No.23 (FIG. 16) of cultured hybridoma, in which thehepatocytes membrane of the portal region was stained in tissue, wasanalyzed on reactivity at hepatocytes surface immediately afterseparation, by using FACS (fluorescence activated cell sorting).Hepatocytes of adult men, 46 years old and 49 years old, obtained bycollagenase perfusion and low-speed centrifugation, were treated withculture supernatant of this sample at 4° C. for 30 minutes, and an FITClabeled anti-mouse IgG antibody was then treated at 4° C. for 30 minutesto make detection by FACS possible. As the result, a part of cells(1-2%) in a hepatocyte population reacted with the sample (FIG. 17). Thereacted cell population, designated as R2 fraction, and the non-reactedcell population, designated as R3 fraction, were fractionated andcultured. Hepatocytes before fractionation were also cultured. As theresult, colony formation was observed on culture after about 7 days inthe hepatocytes before fractionation as described hereinbefore. On theother hand, colony-forming cells were not observed in the R3 fraction,but large numbers of colony were observed in the R2 fraction reactedwith No.23 (FIG. 18). Reactivity with the subcultured human hepatocyteswas examined by FACS, and about 80% of the cells were found to bepositive (FIG. 19). Namely, it was considered that among the subculturedhuman hepatocytes, the differentiated cells during culturing processwere not recognized and only the proliferative hepatocytes wererecognized. From these results, No.23 was suggested to contain hybridomawhich specifically recognized colony-forming cells. Clones obtained bycloning from the No.23 sample were analyzed by using FACS on reactivityat hepatocytes surface immediately after separation. As the result, 3clones showing similar reactivity were obtained. Among these clones, 1clone (Mouse-Mouse hybridoma K8233) was deposited in The InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology (Deposition No. FERM P-18752) on Mar. 6, 2002,and was subjected to international deposition on Mar. 20, 2003(Deposition No. FERM BP-8334).

6. Preparation of a Monoclonal Antibody

The above hybridoma cell (K8223 strain) was cultured, followed byintraperitoneal injection in mice and collection of a monoclonalantibody from ascites to obtain the monoclonal antibody whichspecifically binds to proliferative human hepatocytes.

Example 7 Preparation of a Monoclonal Antibody which SpecificallyRecognizes Human Hepatocytes

A hybridoma producing a monoclonal antibody, which specificallyrecognizes human hepatocytes but does not recognize non-humanhepatocytes, was selected from 88 ELISA-positive samples among 94samples selected at the 1st screening of Example 6 by means ofhistological staining of a human liver tissue. As to 3 samples ofculture supernatant of hybridoma which stained cell membrane ofhepatocytes equally irrespective of the hepatic zones, reactivity tocell surface of cultured human hepatocytes, human hepatocytes just afterseparation and hepatocytes of non-human animals (such as mouse and rat)just after separation was analyzed using FACS. The hepatocytes obtainedfrom a liver of adult men (46 or 68 years old) by collagenase perfusionand low-speed centrifugation were treated with the culture supernatantof the above selected 3 samples at 4° C. for 30 minutes, and thentreated with an FITC-labeled anti-mouse IgG antibody at 4° C. for 30minutes to make detection by FACS possible. As the result of screeningby FACS, 1 sample of hybridoma (pre-cloning) which was reactive withonly human hepatocytes and not reactive with hepatocytes of mouse andrat was selected. In 2 clones of a monoclonal hybridoma obtained fromthe above selected hybridoma, each culture supernatant showed similarreactivity (FIG. 20). In histological staining of the culturesupernatant of the above selected hybridoma, the region considered to bea bile canaliculi of a human liver tissue was stained, but nozone-specificity in terms of staining properties was confirmed. By theway, the liver tissues of a mouse and a rat were both negative onreactivity with the culture supernatant of the hybridoma. Inconsequence, the hybridoma that produces an anti-human hepatocytesmonoclonal antibody, which specifically recognizes human hepatocytes anddoes not recognize non-human hepatocytes, was obtained. This hybridomaK8216 strain was deposited to The Patent Creature Deposition Center ofThe National Institute of Advanced Industrial Science and Technology inJapan as of Mar. 6, 2002 (deposition number: FERM P-18751), and alsodeposited to The International Depository Center as of Mar. 20, 2003(deposition number: FERM BP-8333).

A monoclonal antibody was obtained from the K8216 strain by similarprocedures as described in Example 6.

INDUSTRIAL APPLICABILITY

By the present invention, as described above in detail, proliferation ofhuman hepatocytes in large scale with keeping its original function canbe attained. Furthermore, a chimeric mouse in which many cells in theliver are replaced with human hepatocytes is provided. Drug metabolismtests and safety tests of compounds can be available by the use of saidchimeric mouse or human hepatocytes.

1. A method for proliferating human hepatocytes, which comprisestransplanting human hepatocytes into a liver of an immunodeficienthepatopathy mouse, and then feeding the mouse transplanted with thehuman hepatocytes under such a condition as being protected from theattack by human complement produced by the human hepatocytes therebyproliferating the transplanted human hepatocytes in the mouse liver. 2.The method for proliferating the human hepatocytes of claim 1, whereinthe condition of being protected from the attack by human complement isat least one of the following (a) and (b): (a) the mouse transplantedwith the human hepatocytes is administered at least once with acomplement inhibitor; (b) a progeniture mouse obtained by mating betweenan immunodeficient hepatopathy mouse and a decay-accelerating factor(DAF/CD55) transgenic mouse is utilized as the immunodeficienthepatopathy mouse.
 3. The method for proliferating human hepatocytes ofclaim 1 or 2, wherein the immunodeficient hepatopathy mouse is theprogeniture mouse obtained by mating between a geneticallyimmunodeficient mouse and a genetically hepatopathy mouse.
 4. The methodfor proliferating human hepatocytes of claim 3, wherein the progenituremouse is a hemizygous immunodeficient hepatopathy mouse.
 5. The methodfor proliferating human hepatocytes of claim 4, wherein the hemizygousimmunodeficient hepatopathy mouse is administered with a hepatocytegrowth inhibitor and then the human hepatocytes are transplantedtherein.
 6. The method for proliferating human hepatocytes of any one ofclaims 1 to 5, wherein the immunodeficient hepatopathy mousetransplanted with the human hepatocytes is administered with ananti-mouse Fas antibody.
 7. The method for proliferating humanhepatocytes of claim 1, wherein the human hepatocytes to be transplantedinto the immunodeficient hepatopathy mouse are proliferative humanhepatocytes.
 8. The method for proliferating human hepatocytes of claim7, wherein the proliferative human hepatocytes are human hepatocytesrecognized by a monoclonal antibody which specifically recognizes humanhepatocytes which proliferate with forming colony.
 9. The method forproliferating the human hepatocytes of claim 8, wherein the monoclonalantibody is one produced from Mouse-Mouse hybridoma K8223 (FERMBP-8334).
 10. A method for proliferating human hepatocytes in largescale, which comprises the following steps (1) to (3), and the steps (2)and (3) are repeated at least once; (1) a step comprising transplantinghuman hepatocytes into a liver of an immunodeficient hepatopathy mouse,and then feeding the mouse transplanted with human hepatocytes undersuch a condition as being protected from the attack by human complementproduced by the human hepatocytes thereby proliferating the transplantedhuman hepatocytes in the mouse liver; (2) a step isolating theproliferated human hepatocytes from the mouse liver; and (3) a stepcomprising transplanting the human hepatocytes isolated from the mouseliver into the liver of an immunodeficient hepatopathy mouse, and thenfeeding the mouse transplanted with the human hepatocytes for notshorter than 50 days under such a condition as being protected from theattack by human complement produced by the human hepatocytes.
 11. Themethod for proliferating human hepatocytes in large scale of claim 10,wherein the condition of being protected from the attack by humancomplement in the step (1) and/or the step (3) is at least one of thefollowing (a) and (b): (a) the mouse transplanted with human hepatocytesis administered at least once with a complement inhibitor; (b) aprogeniture mouse obtained by mating between an immunodeficienthepatopathy mouse and a decay-accelerating factor (DAF/CD55) transgenicmouse is utilized as the immunodeficient hepatopathy mouse.
 12. Themethod for proliferating human hepatocytes in large scale of claim 10 or11, wherein the immunodeficient hepatopathy mouse in the step (1) and/orthe step (3) is the progeniture mouse obtained by mating between agenetically immunodeficient mouse and a genetically hepatopathy mouse.13. The method for proliferating human hepatocytes in large scale ofclaim 12, wherein the progeniture mouse is a hemizygous immunodeficienthepatopathy mouse.
 14. The method for proliferating human hepatocytes inlarge scale of claim 13, wherein the hemizygous immunodeficienthepatopathy mouse is administered with a hepatocyte growth inhibitor andthen human hepatocytes are transplanted therein.
 15. The method forproliferating human hepatocytes in large scale of any one of claims 10to 14, wherein the immunodeficient hepatopathy mouse transplanted withhuman hepatocytes in the step (1) and/or the step (3) is administeredwith an anti-mouse Fas antibody.
 16. The method for proliferating humanhepatocytes in large scale of claim 10, wherein the human hepatocytes tobe transplanted into the liver of the immunodeficient hepatopathy mousein the step (1) and/or the step (3) are proliferative human hepatocytes.17. The method for proliferating human hepatocytes in large scale ofclaim 16, wherein the proliferative human hepatocytes are humanhepatocytes recognized by a monoclonal antibody which specificallyrecognizes human hepatocytes which proliferate with forming colony. 18.The method for proliferating human hepatocytes in large scale of claim17, wherein the monoclonal antibody is one produced from Mouse-Mousehybridoma K8223 (FERM BP-8334).
 19. The method for proliferating humanhepatocytes in large scale of claim 10, wherein only human hepatocytesare substantially isolated in step (2) by at least one of the followingprocedures (a) and (b); (a) to treat a liver tissue separated from themouse liver with collagenase; and (b) to isolate cells being recognizedby a monoclonal antibody which specifically recognizes human hepatocytesbut not recognizes non-human hepatocytes.
 20. The method forproliferating human hepatocytes in large scale of claim 19, wherein themonoclonal antibody is one produced by Mouse-Mouse hybridoma K8216 (FERMBP-8333).
 21. A chimeric mouse carrying in the liver human hepatocytesproliferated by the method for proliferating human hepatocytes of anyone of claims 1 to 9, or by the method for proliferating humanhepatocytes in large scale of any one of claims 10 to
 20. 22. Thechimeric mouse of claim 21, wherein the proliferated human hepatocytesmake up not less than 70% of the cells in the liver.
 23. The chimericmouse of claims 21 or 22, wherein said mouse has human-type P450activity.
 24. A method for obtaining human hepatocytes comprisesisolating the human hepatocytes from the liver of the chimeric mouse ofclaims 21, 22 or
 23. 25. The method for obtaining human hepatocytes ofclaim 24, wherein only human hepatocytes are substantially isolated byat least one of the following procedures (a) and (b); (a) to treat aliver tissue separated from the mouse liver with collagenase; and (b) toisolate cells being recognized by a monoclonal antibody whichspecifically recognizes human hepatocytes but not recognizes non-humanhepatocytes.
 26. The method for obtaining human hepatocytes of claim 25,wherein the monoclonal antibody is one produced from Mouse-Mousehybridoma K8216 (FERM BP-8333).
 27. Human hepatocytes obtained by themethod of any one of claims 24 to
 26. 28. A cellular kit containing thehuman hepatocytes of claim
 27. 29. A hybrid-type artificial liver filledwith the human hepatocytes of claim
 27. 30. A monoclonal antibody whichspecifically recognizes human hepatocytes but not recognize non-humanhepatocytes.
 31. The monoclonal antibody of claim 30, which is oneproduced from Mouse-Mouse hybridoma K8216 (FERM BP-8333). 32.Mouse-Mouse hybridoma K8216 (FERM BP-8333).
 33. A method for testingpharmaceutical kinetics or toxicity of a candidate substance, whichcomprises systemically administering the substance into the chimericmouse of claims 21, 22 or 23.